Method for detecting a target nucleic acid sequence

ABSTRACT

A method of detecting a target nucleic acid sequence comprising providing a stem-and-loop structured nucleic acid for measurement wherein the nucleic acid comprises complementary sequence portions located at both terminals and a target sequence portion therebetween as well as a double-stranded portion formed by hybridization of the complementary sequence portions located at both terminals and a remaining looped single-stranded portion, providing a probe nucleic acid having a sequence complementary to the target sequence portion wherein one end of the probe nucleic acid being immobilized to a solid substrate surface, reacting the nucleic acid for measurement with the probe nucleic acid to specifically hybridize the target sequence portion of the nucleic acid for measurement to the probe nucleic acid, and detecting presence or absence of the nucleic acid for measurement hybridized to the probe nucleic acid.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a technique for detecting a targetnucleic acid, more particularly to a method for detecting a targetnucleic acid sequence present in a LAMP amplified product and a LAMPamplified product for use in detection of a target nucleic acid.

2. Description of the Related Art

To select correct therapeutic agents, it is important to identifybacteria causing a given infection. Currently, culture method andnucleic acid amplification method are used for microorganism tests.

Recently, improvement in culture method has been made in an effort toimprove detection sensitivity and to reduce days of culture.Immunochromatography utilizing antigen-antibody reactions rapidlybecomes popular as a simple identification method (see, for example,Japanese Patent Publication No. 2001-103981 (paragraph 0011-0012) andJapanese Patent Publication No. 2002-125695 (paragraph 0002)). However,since this method still takes several weeks for bacterial growth, ithardly meets the needs sought in clinical settings. Consequently,patients are often given a wrong treatment until identification resultsare obtained.

In contrast, the nucleic acid amplification method uses specific primersto identify bacterial species or resistant bacteria by investigatingpresence or absence of amplification. This method, including a samplepreparation step, can provide test results within approximately 6-7hours, making it a very useful rapid test. Furthermore, under the recentgenomic analysis competition, entire genetic information of manyorganisms are being analyzed, and as a result, this method is expectedto become available for identification of a wide range of bacterialspecies.

A method well-known as a nucleic acid amplification technique is PCRmethod (Polymerase Chain Reaction method, Roche). PCR method is widelyused as a tool for genetic analyses such as gene cloning and structuraldetermination. However, PCR method has a disadvantage of requiring acomplex temperature control device like Thermal Cycler and reaction timeover two hours. In PCR method, If synthesis of wrong complementarychains occurs by any chance, the resultant products work as templatesfor amplification, thereby leading to a wrong identification. In fact,it is difficult to regulate specific amplification based on a differenceof only one base in a primer terminal.

In addition, due to the fact that double-stranded products are generatedin general when target gene products amplified by PCR method aredetected by a DNA chip, complementary strands work as competitors forprobes upon a hybridization reaction with the probes, reducinghybridization efficiency and detection sensitivity. To address thisproblem, such methods as digesting or separating complementary strandshave been employed to turn a target into a single-stranded sequence.However, this method has still several problems including a need to useenzymes, expensiveness due to the use of magnetic beads, and handlingcomplexity.

LAMP method (Loop-mediated isothermal amplification method) has beendeveloped as a gene amplification method to fix this problem. LAMPmethod completes gene amplification within one hour under an isothermalcondition. Also, 100-1,000 times amount of final products are obtainedcompared to that of PCR products. LAMP method also has an advantage ofhigher specificity as compared to PCR method because six primer regionsare set in LAMP method (see, for example, U.S. Pat. No. 3,313,358). LAMPmethod is expected to be a promising technique to rapidly detectbacteria, viruses and gene mutations (see, for example, Japanese PatentPublication No. 2003-159100) with high sensitivity.

Among tests using LAMP method, a method is now commercialized whichdetects presence or absence of gene amplification by measuring whiteturbidity of magnesium pyrophosphate, a by-product which is produced inthe course of amplification (see, for example, Japanese PatentPublication No. 2003-174900). In this method where white turbidity of aby-product (magnesium pyrophosphate) is measured, there is no way ofconfirming whether amplification of an unintended product occurs or not.LAMP method has also a problem that it cannot detect multiple targetgenes simultaneously.

In addition, methods using intercalators or optical properties have beenknown as procedures for detecting LAMP amplified products (see, forexample, Japanese Patent Publication No. 2002-186481). Other knownmethods are those measuring degree of fluorescence polarization ofreaction solution by fluorescently labeling a probe which hybridizes toa single-stranded loop portion present in a LAMP product (see, forexample, Japanese Patent Publication 2002-272475) as well as thoseimmobilizing an insoluble carrier to a 5′ terminal side of a primerwhich hybridizes to a single-stranded loop portion and observing anaggregation reaction associated with amplification reaction (see, forexample, Japanese Patent Publication 2002-345499).

BRIEF SUMMARY OF THE INVENTION

According to embodiments of the present invention, there is provided amethod of detecting a target nucleic acid sequence comprising:

providing a stem-and-loop structured nucleic acid for measurementwherein the nucleic acid comprises complementary sequence portionslocated at both terminals and a target sequence portion therebetween aswell as a double-stranded portion formed by hybridization of thecomplementary sequence portions located at both terminals and aremaining looped single-stranded portion;

providing a probe nucleic acid having a sequence complementary to thetarget sequence portion wherein one end of the probe nucleic acid beingimmobilized to a solid substrate surface;

reacting the nucleic acid for measurement with the probe nucleic acid tospecifically hybridize the target sequence portion of the nucleic acidfor measurement to the probe nucleic acid; and

detecting presence or absence of the nucleic acid for measurementhybridized to the probe nucleic acid.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

FIG. 1 is a schematic diagram illustrating an amplification method usinga conventional LAMP method.

FIG. 2 is a schematic diagram illustrating an amplification productobtained by a conventional LAMP method.

FIG. 3 is a schematic diagram illustrating an amplification method usedfor production of the nucleic acid for measurement according to anembodiment of the present invention.

FIG. 4 is a schematic diagram illustrating the nucleic acid formeasurement according to an embodiment of the present invention.

FIG. 5 schematically illustrates hybridization reaction between nucleicacid probe and a LAMP product in which a target sequence is located inits looped single stranded portion and a nucleic acid probe. In FIGS. 5Aand 5B, 5′ to 3′ sequence orientation of both the nucleic acid probe andthe target sequence portion are arranged in a manner where thedouble-stranded portion in the LAMP product extends away from thesubstrate plate. In contrast, in FIG. 5C, the 5′ to 3′ sequenceorientation is arranged in a manner where the double stranded portionextends toward the substrate plate.

FIG. 6 schematically illustrates hybridization reaction between nucleicacid probe and a LAMP product in which a target sequence is located inits looped single stranded portion. While, in FIG. 6A, thedouble-stranded portion in the LAMP product extends away form thesubstrate plate, in FIG. 6B, the double-stranded portion extends towardthe substrate plate.

FIG. 7 shows a primer which is designed to have a target nucleic acid ina double-stranded region (stem region) as well as the position of atarget sequence in NAT2 gene.

FIG. 8 shows a primer which is designed to have a target nucleic acid ina single-stranded region (loop region) as well as the position of atarget sequence in NAT2 gene.

FIG. 9 is an electrophoresis of LAMP amplification products designed tohave a target nucleic acid in a double-stranded region (stem region) ora single-stranded region (loop region) and an electrophoresis ofproducts treated by restriction enzymes.

FIG. 10 shows an arrangement of electrodes used in the Examples todetect LAMP products.

FIG. 11 is graphs showing measurement results of electrical detection ofLAMP products performed in the Example. FIG. 11A is a graph showingmeasurement results obtained from LAMP products having a target nucleicacid in its double-stranded portion, and FIG. 11B is a graph showingmeasurement results obtained from LAMP products having a target nucleicacid in a looped single-stranded portion.

FIG. 12 illustrates an arrangement of electrodes used in the Example 2to detect SNPs in LAMP products.

FIG. 13 is graphs showing measurement results of electrical detection ofSNPs in LAMP products performed in the Example 2. FIG. 13A is a graphshowing measurement results obtained from substrate plates washedquickly with ultrapure water after hydridization. FIG. 13B is a graphshowing measurement results obtained from substrate plates immersed inwashing buffer at 35° C. for 40 minutes after hybridization. FIG. 13C isa graph showing measurement results obtained from substrate platesimmersed in washing buffer at 40° C. for 40 minutes after hybridization.FIG. 13D is a graph showing measurement results obtained from substrateplates immersed in washing buffer at 45° C. for 40 minutes afterhybridization.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the present invention will now be described in moredetail.

<Nucleic Acids for Measurement>

Now, primer designing in LAMP method and intermediate products servingas origins of amplification will be described with reference to FIG. 1and FIG. 2. In LAMP method, six primer regions will be set and fourprimers are used for gene amplification. FIG. 1 illustratesdouble-stranded DNA to be detected. Three regions, F3 region, F2 regionand F1 region, in order of proximity to 5′ terminal of the doublestrand, will be determined and three regions, B3c region, B2c region andB1c region, in order of proximity to 3′ terminal, will be determined. Inaddition, F3 region, F2 region and B1 region in complementary strandthereof are called F3c region, F2c region and F1c region, and B3cregion, B2c region and B1c regions in complementary strand thereof arecalled B3 region, B2 region and B1 region, respectively. The sixregions, F3 region, F2 region, F1 region, B3c region, B2c region, B1cregion and complementary strands regions thereto are hereinafterdesignated as a primer designing region. Primers constituting four basicprimers are FIP primer that has a same sequence with the F2 region atits 3′ terminal and a sequence complementary to the F1 region at its 5′terminal, F3 primer comprised of a same sequence with the F3 region, BIPprimer having a sequence complementary to B2c region at its 3′ terminaland a same sequence with the B1c region at its 5′ terminal, and B3primer comprised of a sequence complementary to the B3c region. LAMPamplification using the four primers described above will causeformation of a dumbbell-shaped intermediate product having astem-and-loop structure as shown in FIG. 2. Having sequencescomplementary to each other in the same nucleic acid strand, bothterminal portions of the intermediate product will self-anneal and forma single-stranded loop. Intermediate product generation and subsequentamplification process have been well known and are described in detailin Japanese Patent 3,313,358 and Japanese Patent Publication2002-186481. Traditionally, in LAMP amplification products, targetsequences are interposed between B1 and F1c or between F1 and B1c.

In contrast to traditional target sequences shown in FIG. 1 and FIG. 2,primers used in this invention are designed to have target sequenceslocated in the single-stranded loop portion. Accordingly, in thisinvention, target sequence regions are designed to be located eitherbetween primer regions F1c and F2c, primer regions F2 and F1, primersB1c and B2c or primer regions B2 and B1 as shown in FIG. 3. Theseregions are designated as FPc region, FP region, BPc region and BPregion, respectively. Location of target sequence designs are shown inFIG. 3. Four kinds of primers will be designed based on the six primerregions designed in this way. LAMP amplification using these primerswill provide portions of LAMP amplification products as shown in FIG. 4.In these LAMP amplification products, target sequence regions; FPcregion, FP region, BP region and BPc region will be located in asingle-stranded loop of amplified products. Since F1 region and F1cregion, and B1 region and B1c region have complementary sequences fromthe beginning, they will self-hybridize with each other to give doublestrands. In this case, F2c region, F2 region, B1c region and B1 regionwill be located in loop regions of single strands as shown in FIG. 4.These regions, F2c region, F2 region, B1c region and B1c region, mayoverlap, in part, with target sequence regions FPc region, FP region,BPc region and BP region. Since some target sequences included inamplification products are single-stranded as shown in FIG. 4, they canbe detected, without any denaturation procedure, by specifichybridization with probe nucleic acids FP, FPc, BP and BPc that arecomplement to each target sequences as shown in FIG. 4. As used herein,specific hybridization means that it can detect minor differences whenSingle Nucleotide Polymorphisms (SNPs) or mutations occur.

<Probe Nucleic Acids>

As explained in FIG. 4 according to the present invention, targetsequences included in single-stranded loop structures of stem-and-loopstructured nucleic acids for measurement are detected by means of probenucleic acids having sequences complementary thereto. These probenucleic acids are immobilized onto a solid substrate surface. Probes areused typically comprising a part of a DNA chip.

A DNA chip is a several-centimeter-square of glass or silicon substrateonto which several tens to several hundred thousand kinds probes withdifferent sequences are immobilized. DNA chips allow for simultaneousinvestigation of information on multiple sequences. This enablesanalyses of gene expression patterns and SNPs to be completed in severaldays, which required several weeks previously. Currently, DNA chips aremainly used in search for new genes, elucidation of functions andtechniques for supporting researches, but recently, they are becomingcommon techniques for disease diagnosis.

As an exemplary DNA chip, a technique of AFFYMETRIX are well known (see,for example, Proc. Natl. Acad. Sci. USA 91, 1994, p 5022-5026). In thistechnique, fluorescently-labeled sample genes are reacted with probes ona chip and detected by means of a high-sensitive fluorescent analysisapparatus. Another type of a detection method developed is a currentdetection type DNA chip. In this method, intercalators specificallyreactive with double-stranded DNA are added and electrochemical signalsgenerated by the intercalators are measured. Electrical DNA chips areexpected to be a promising second-generation DNA chip, because they needno labeling and expensive detection apparatuses (see, for example,Japanese Patent Publication H05-199898).

<Hybridization Reaction between Nucleic Acids for Measurement andProbes>

The nucleic acids for measurement are bound to the solid surface viaspecific hybridization reaction between single-stranded target sequencelocated therein and the probe nucleic acids. In this invention, it is anadditional characteristics that 5′ to 3′ sequence orientations of boththe probe nucleic acid and the target sequence portion are arranged sothat the double-stranded portion of the nucleic acid for measurementextends away from the solid surface when the probe nucleic acid and thetarget sequence portion are hybridized (see FIG. 5A). Suchcharacteristics have been found based on a finding that if 5′ to 3′sequence orientations of both the probe nucleic acid and the targetsequence portion are arranged as shown in FIG. 5C, complex conformationscharacteristics to LAMP products cause steric hindrance together with aprobe-bound substrate (see FIG. 6B) and this causes reduction inhybridization efficiency. Steric hindrance produced by LAMP products anda solid substrate on which probe nucleic acids are bound can be avoided(see FIG. 6A) by arranging 5′ to 3′ sequence orientations of both theprobe nucleic acid and the target sequence portion so that thedouble-stranded portion of the nucleic acid for measurement extends awayfrom the solid surface when the probe nucleic acid and the targetsequence portion are hybridized (see FIG. 5B), thereby achievingimprovement in hybridization efficiency.

<Detection of Nucleic Acids for Measurement Hybridized to Probe NucleicAcids>

In this invention, when detecting presence or absence of the nucleicacids for measurement hybridized to probe nucleic acids, detection meansis not limited to particular means in any way. For example, detectioncan be made based on fluorescent labels or by means of electricaldetection using a double-strand specific intercalator generatingelectrical potential

EXAMPLE 1

A method for nucleic acid detection according to an embodiment of thepresent invention will now be described more specifically by way of theExamples.

In this example, LAMP amplification products are produced as a samplenucleic acid and target nucleic acid(s) present in the LAMPamplification products were detected in an electrical current systemafter hybridization reaction. For LAMP reaction, two sets of thefollowing primers were used and a part of N-Acetyltransferase 2 (NAT2)gene were amplified

(1) Synthetic Oligonucleotide

Primer 1

Target nucleic acid sequences were designed to be located in adouble-stranded region (stem region) of the LAMP amplification products.Peripheral genomic sequences are shown in FIG. 7 (SEQ ID NO: 15).

NAT F3 Primer: 5′ ACAGAAGAGAGAGGAATCTGGT 3′ (SEQ ID NO: 1) NAT FIPPrimer: 5′ TGTTTCTTCTTTGGCAGGAGATGAGAA-GGACCAAATCAGGAGAGAGCA 3′ (SEQ IDNO: 2) NAT B3 Primer: 5′ GATGAAGCCCACCAAACAGTA 3′ (SEQ ID NO: 3) NAT BIPPrimer: 5′ ATGAATACATACAGACGTCTCCCTGGGGTCTGCAAGGAAC 3′ (SEQ ID NO: 4)

Primer 2

Target nucleic acid sequences were designed to be located in asingle-stranded region (loop region) of the LAMP amplification products.Peripheral genomic sequences are shown in FIG. 8 (SEQ ID NO: 15)

NAT F3 Primer: 5′ ACAAACAAAGAATTTCTTAATTCTCAT 3′ (SEQ ID NO: 5) NAT FIPPrimer: 5′ CGTCTGCAGGTATGTATTCATAGACTCAAAAAATATACTTATTTACGCTTGAACC 3′(SEQ ID NO: 6) NAT B3 Primer: 5′ CGACCAGATCTGTATTGTCTT 3′ (SEQ ID NO: 7)NAT BIP Primer: 5′ ATAACCACATCATTTTGTTCCTTGCATGAATTTTCTATAGGTGAGGATGA 3′(SEQ ID NO: 8)

(2) LAMP Reaction Solution

The composition of the LAMP reaction solution was as follows:

Sterile ultrapure water 1.5 μL Bst DNA polymerase 1 μL buffer 12.5 μLbuffer components Tris HCl (pH 8.0) 40 mM KCl 20 mM MgSO₄ 16 mM(NH₄)₂SO₄ 20 mM Tween 20 0.2% Betaine 1.6 M dNTP 2.8 mM F3-primer (10μM) 0.5 μL B3-primer (10 μM) 0.5 μL FIP-primer (10 μM) 4 μL BIP-primer(10 μM) 4 μL Template (purified human genome) 1 μL Total 25 μL

(3) Nucleic Acid Amplification by LAMP Method

Nucleic acid amplification was performed at 58° C. for one hour. Fornegative control, sterile water was added instead of templates.

(4) Confirmation of Nucleic Acid Amplification

LAMP products amplified by the methods described above were confirmed byagarose-gel electrophoresis as shown in FIG. 9. The presence or absenceof products of interest were confirmed by restriction enzyme cleavage.In FIG. 9, lanes 1-7 correspond to the following samples 1-7,respectively.

Electrophoretic results are shown below.

-   -   1. 100 bP ladder (TAKARA)    -   2. Negative control: a target nucleic acid is located in a        double-stranded region    -   3. Positive control: a target nucleic acid is located in a        double-stranded region    -   4. Negative control: a target nucleic acid is located in a        single-stranded region    -   5. Positive control: a target nucleic acid is located in a        single-stranded region    -   6. Pst I restriction enzyme treated: a target nucleic acid is        located in a double-stranded region    -   7. Pst I restriction enzyme treated: a target nucleic acid is        located in a single-stranded region

Digestion with a restriction enzyme gave fragments as expected bytheory. This indicates these LAMP products are specific amplificationproducts.

(5) Preparation of Nucleic Acid Probe Immobilized Electrodes

Nucleotide sequences of nucleic acid probes are shown below.

-   -   Positive-probe FP: AACCTCGAACAATTG (SEQ ID NO: 9) 3′ SH, 5′ SH        probes total of two    -   Positive-prove FPc: CAATTGTTCGAGGTT (SEQ ID NO: 10) 3′ SH, 5′ SH        probes total of two    -   N-probe NP: CTGGACGAAGACTGA (SEQ ID NO: 11)

FP probe and FPc probe are complementary to each other. A total of four3′ SH and 5′ SH modified probes were tested for FP probe and FPc probe.In contrast, N-probe served as negative control and had sequencesunrelated to four probes described above.

Probe solution containing the labeled probes was spotted onto goldelectrodes and, after standing one hour, the electrodes were immersed inmercaptohexanol solution and washed 0.2×SSC solution. The electrode werethen washed with ultrapure water, air-dried and used asprobe-immobilized electrodes. Arrangement of electrodes is as shown inFIG. 10.

(6) Hybridization of LAMP Products to Nucleic Acid Probes

LAMP products amplified in the step (3) above were used as samplenucleic acids. The surface prepared in step (4) on which nucleic acidprobes were immobilized was immersed in LAMP products added by 2×SSCsalt, hybridization reaction was performed by standing at 35° C. Theprobe-immobilized surface was quickly washed with ultra pure water. Theelectrodes were immersed for 15 minutes in phosphate buffer containing50 μM Hoechst 33258 solution (an intercalator) and oxidation currentresponse of Hoechst 33258 molecule was measured.

(7) Results

In FIG. 11, the results of electrical current measurements were shown asan increment of current values generated in electrodes on which eachprobes were immobilized. In LAMP products in which target sequences arelocated in a double-stranded portion (stem portion), no increment incurrent values derived from hybridization was found for NP, 3′ SH FP, 3′SH FPc, 5′ SH FP and 5′ SH FPc (FIG. 11A). In contrast, in LAMP productsin which target nucleic acid is located in a single-stranded loopportion, no increment in current values derived from hybridization wasfound for NP, 3′ SH FP and 5′ SH FP, but increment in current valueprobably derived from hybridization was found for 3′ SH FPc and 5′ SH FP(FIG. 11B).

For 3′ SH FP and 5′ SH FPc, which gave no current signal values increasewhen used with products having target nucleic acid located in asingle-stranded loop portion, the double-stranded portion in LAMPproducts, upon hybridization with the single-stranded loop portion ofLAMP products, extended toward the substrate thereby causing sterichindrance (see FIG. 5C and FIG. 6B). In contrast, for 3′ SH FPc and 5′SH FP, which gave current signal value increase, the double-strandedportion in LAMP products extends away from the substrate thereby streichindrance is avoided (see FIG. 5B and FIG. 6A).

It was found, from these results, that it was essential to locate targetnucleic acids in looped single-stranded portions to detect LAMP productswith DNA chips. It was also found that 5′ to 3′ sequence orientations ofboth the probe nucleic acid and the target sequence portion must bearranged so that the double-stranded portion of LAMP products extendedaway from the solid surface in order to avoid steric hindrance occurredbetween LAMP products and substrates upon hybridization reaction.

EXAMPLE 2

By way of an application of a nucleic acid detection method according tothis invention, single nucleotide polymorphisms (SNPs) in target nucleicacid sequences were detected.

In Example 2, LAMP products were prepared for sample nucleic acids asdescribed in Example 1. After hybridization, SNPs in the target nucleicacid sequences in the LAMP products were detected in a electricalcurrent detection system.

(1) LAMP Products Used for Detection

LAMP products used were same as those amplified by Primer 2 described inExample 1, (1) Synthetic oligonucleotide.

(2) Probes used for detection

Nucleotide sequences of the nucleic acid probes are shown below

Positive-probe FPc: CAATTGTTCGAGGTT 3′SH (a probe used in Example 1)(SEQ ID NO: 10) Positive-probe FPc SNP: CAATTGTTGGAGGTT 3′ SH (SEQ IDNO: 12) Positive-probe FPc 25mer: ATCTTCAATTGTTCGAGGTTCAAGC 3′ SH (SEQID NO: 13) Positive-probe FPc 25mer SNP: ATCTTCAATTGTTGGAGGTTCAAGC 3′ SH(SEQ ID NO: 14) N-probe NP: CTGGACGAAGACTGA 3′ SH (SEQ ID NO: 11)

FPc and NP were same as those used in Example 1. For FPc SNP, G near thecenter of FPc sequence was changed into C. FPc 25 mer was a targetnucleic acid sequence having five base extensions in both upstream anddownstream of FPc. For FPc 25 mer SNP, G near the center of FPc 25 mersequence was changed into C. All five probes above were 3′ SH modified.

Probes were immobilized in the same manner as described in theExample 1. Arrangement of electrodes is shown in FIG. 12.

(3) Hybridization of LAMP Products to Nucleic Acids

LAMP products amplified in the above step (1) were used as samplenucleic acids. The surface prepared in step (2) on which nucleic acidprobes were immobilized was immersed in LAMP products added by 2×SSCsalt, and by standing for 60 minutes at 35° C., hybridization reactionwas performed. The substrates washed under four different conditionswere made: immersed in 0.2×SSC buffer at 35° C., 40° C. or 45° C. for 40minutes followed by quick wash with ultra pure water; or just quicklywashed with ultrapure water after hybridization. The electrodes wereimmersed for 15 minutes in phosphate buffer containing 50 μM Hoechst33258 solution (an intercalator) and oxidation current response ofHoechst 33258 molecule was measured.

(4) Results

In FIG. 13, the results of electrical current measurements were shown asan increment of electrical current generated in electrodes on which eachprobes had been immobilized. Regarding the FPc and FPcSNP, (A) there wasno increase in electrical current values for FPcSNP when used with asubstrate quickly washed with ultrapure water after hybridization, onthe contrary, significant increase in electrical current values wasobserved for FPc. In addition, (B) increase in electrical current valueswas disappeared for both FPc and FPc SNP when used with substrates thathad been washed with 0.2×SSC solution at 35° C. for 40 minutes afterhybridization. This indicates FPc and FPc SNP can identify SNPs underthe washing condition (A).

Regarding extended target sequences, FPc 25 mer and FPc 25 mer SNP, (C)increase in electrical current values was found for both FPc 25 mer andFPc 25 mer SNPs when used with substrates that had been washed with0.2×SSC solution at 40° C. for 40 minutes after hybridization. Incontrast, (D) when substrates had been washed with 0.2×SSC solution at45° C. for 40 minutes after hybridization, no increase in electricalcurrent values was found for FPc 25 mer SNP, but significant increasewas found for FPc 25 mer. This indicates FPc 25 mer and FPc 25 mer SNPcan identify SNPs under the washing condition (D).

These results show that SNPs can be detected by selecting appropriatehybridization or washing conditions depending on different probesequences, or by selecting optimal probes for defined hybiridization andwashing condition.

This invention shall never be limited to the embodiments specificallydescribed above and, in practicing this invention, constituent elementsthereof can be modified to give other embodiments without departing fromthe spirit or scope of the invention. In addition, various inventionswill be given by combining multiple constituent elements disclosed inthe embodiment stated above. For example, among all constituent elementsmentioned in the embodiments, some constituent elements could beomitted. Furthermore, constituents elements of different embodimentscould appropriately combined.

Additional advantages and modifications will readily occur to thoseskilled in the art. Therefore, the invention in its broader aspects isnot limited to the specific details and representative embodiments shownand described herein. Accordingly, various modifications may be madewithout departing from the spirit or scope of the general inventiveconcept as defined by the appended claims and their equivalents.

1. A method of detecting a target nucleic acid sequence comprising:providing a stem-and-loop structured nucleic acid for measurementwherein the nucleic acid comprises complementary sequence portionslocated at both terminals and a target sequence portion therebetween aswell as a double-stranded portion formed by hybridization of thecomplementary sequence portions located at both terminals and aremaining looped single-stranded portion; providing a probe nucleic acidhaving a sequence complementary to the target sequence portion whereinone end of the probe nucleic acid being immobilized to a solid substratesurface; reacting the nucleic acid for measurement with the probenucleic acid to specifically hybridize the target sequence portion ofthe nucleic acid for measurement to the probe nucleic acid; anddetecting presence or absence of the nucleic acid for measurementhybridized to the probe nucleic acid by using a double strand specificintercalator, thereby detecting the target nucleic acid sequence.
 2. Amethod according to claim 1, wherein 5′ to 3′ sequence orientations ofboth the probe nucleic acid and the target sequence portion are arrangedso that the double-stranded portion of the nucleic acid for measurementextends away from the solid surface when the probe nucleic acid and thetarget sequence portion is hybridized.
 3. A method according to claim 1,wherein the nucleic acid for measurement and the probe nucleic acid areDNA.
 4. A method according to claim 2, wherein the nucleic acid formeasurement and the probe nucleic acid are DNA.
 5. A method according toclaim 1, wherein the probe nucleic acid is a component of a DNA chip. 6.A method according to claim 2, wherein the probe nucleic acid is acomponent of a DNA chip.
 7. A method according to claim 1, wherein thenucleic acid for measurement is a LAMP product amplified by LAMP method,wherein the LAMP product comprises at least one part selected from thegroup consisting of a first part, a second part, a third part, and afourth part of the LAMP product; the first part of the LAMP productcomprising F1c, F2c, and F1 arranged in this order, the second part ofthe LAMP product comprising F1c, F2, and F1 arranged in this order, thethird part of the LAMP product comprising B1c, B2c, and B 1 arranged inthis order, and the fourth part of the LAMP product comprising B1c, B2,and B1 arranged in this order.
 8. A method according to claim 2, whereinthe nucleic acid for measurement is a LAMP product amplified by LAMPmethod, wherein the LAMP product comprises at least one part selectedfrom the group consisting of a first part, a second part, a third part,and a fourth part of the LAMP product; the first part of the LAMPproduct comprising F1c, F2c, and F1 arranged in this order, the secondpart of the LAMP product comprising F1c, F2 and F1 arranged in thisorder, the third part of the LAMP product comprising B1c, B2c, and B1arranged in this order, and the fourth part of the LAMP productcomprising B1c, B2, and B1 arranged in this order.
 9. A method accordingto claim 7, wherein the target sequence in the LAMP product is insertedbetween F1 region and F2 region, between F2c region and F1c region,between B 1 region and B2 region, and/or between B2c region and B1cregion of the LAMP product.
 10. A method according to claim 8, whereinthe target sequence in the LAMP product is inserted between F1 regionand F2 region, between F2c region and F1c region, between B1 region andB2 region, and/or between B2c region and B1c region of the LAMP product.11. A method according to claim 9, wherein a part of the target sequenceoverlaps with a part of the sequence of the F2 region, F2c region, B2region, and/or B2c region of the LAMP product.
 12. A method according toclaim 10, wherein a part of the target sequence overlaps with a part ofthe sequence of F2 region, F2c region, B2 region, and/or B2c region ofthe LAMP product.
 13. A method according to claim 1, wherein thepresence or absence of the nucleic acid for measurement hybridized tothe probe nucleic acid is detected electrically using a double-strandspecific intercalator.
 14. A method according to claim 2, wherein thepresence or absence of the nucleic acid for measurement hybridized tothe probe nucleic acid is detected electrically using a double-strandspecific intercalator.